Chronic wound microenvironment mediates selection of biofilm-forming multi drug resistant Staphylococcus epidermidis with capability to impair healing

Venous leg ulcers (VLU) are the most common chronic wounds characterized by bacterial biofilms and perturbed microbiome. Staphylococcus epidermidis is primarily known as skin commensal beneficial for the host, however, some strains can form biofilms and cause infections. By employing shotgun metagenomic sequencing we show that genetic signatures of antimicrobial resistance, adhesion and biofilm formation in VLU isolates correlate with in vitro bacterial traits. We demonstrate that the capability of chronic wound isolates to form biofilms and elicit IL-8 and IL-1β expression in human ex vivo wounds, correlates with the non-healing outcomes in patients with VLU. In contrast, commensal strains were incapable of surviving in the human ex vivo wounds. We show that major fitness traits of S. epidermis from VLU involve genes for resistance to methicillin and mupirocin, while the biofilm formation relied on the minimal number of genetic elements responsible for bacterial binding to fibronectin and fibrinogen. This underscores the importance of the emergence of treatment resistant virulent lineages in patients with non-healing wounds.


Introduction
Venous leg ulcers (VLUs) represent the most common chronic wound affecting millions of patients and leading to billions of dollars of associated healthcare costs annually 1,2 . The chronic ulcer microenvironment is a suitable site for the development of biofilms that can compromise healing and contribute to severe complications 3,4 . While the role of Staphylococcus aureus in chronic wound pathology has been extensively studied [4][5][6] , the contribution of its close relative, Staphylococcus epidermidis, to impaired healing has not yet been evaluated.
As one of the first commensals, S. epidermidis contributes to healthy cutaneous barrier by inhibiting colonization of pathogens and regulating the immune response 7 . S. epidermidis also has an educating role for the variety of resident immune cells, priming them to respond to their pathogenic relatives like S. aureus 8,9 . S. epidermidis can also stimulate keratinocytes to produce broad spectrum antimicrobial peptides (AMP) 10 . Alongside its beneficial role, S. epidermidis could carry a reservoir of antimicrobial resistance (ABR) genes and virulence factors which places this microbe to the list of "accidental" pathogens known to cause nosocomial and deviceassociated infections [11][12][13] . S. epidermidis can carry genes encoding polysaccharide intercellular adhesins (icaADBC) and accumulation-associated protein (aap) linked to biofilm formation, but also fibronectin (embp), fibrinogen (sdrG) and keratin/collagen I (sdrF) binding genes important for adhesion to different surfaces 11 . Biofilms and disturbed microbiota are also a hallmark of nonhealing chronic wounds including VLUs 3,14 . While comparative whole-genome sequencing (WGS) methods have been utilized for determining pathogenicity among disease associated S. epidermidis strains 12 , chronic wound isolates have not been analyzed before.

Results and Discussion
We report the first metagenomic and functional characterization of S. epidermidis isolates from chronic VLU (CW9, CW20 and CW48) and comparison to commensal skin strains (CCN024 and CCN027). Using the WGS we identified prevalence of ABR in VLU strains, not present in commensal isolates, confirmed in MIC assays (Fig. 1a&b). Specifically, mupA and mecA genes and associated resistance to mupirocin and oxacillin/methicillin, respectively, were key features of the VLU isolates CW9 and CW48. In contrast to CW9 and CW48, strain CW20 showed susceptibility to oxacillin and mupirocin. MecA has been associated with S. epidermidis virulence in systemic and device-related infections12, however mupirocin resistance in pathogenic isolates has not been reported before. In addition, higher MIC values for benzalkonium chloride, an antiseptic used in chronic wound management, were detected in VLU isolates but not in commensals, while only CW20 strain harbored the qacA/qacB encoding resistance to quaternary ammonium compounds (Fig. 1a&b). Our findings raise significant concern for VLU patients, as both mupirocin and benzalkonium chloride are often used for preoperative skin decolonization of S. aureus 15  We further evaluated the gene repertoire encoding the components required for binding to host extracellular matrix (ECM) and detected most of these genes in all isolates (Fig. 1c). The expression of embp and sdrG, present in all strains, was evaluated during the exponential and stationary growth (Fig. 1d), revealing significantly higher embp expression in VLU in comparison to healthy skin isolates regardless of the growth phase (Fig. 1e). Expression of sdrG was upregulated in two chronic wound isolates, CW20 and CW48, while in CW9 strain remained comparable to levels in the commensals (Fig 1f). We also detected higher expression of sdrG in commensal CCN024, not sustained at the stationary phase. Altogether, the differences in embp and sdrG expression indicated better colonizing and biofilm forming properties of VLU isolates and correlated with potential of CW20 and CW48 to form biofilm in vitro, while CW9 showed higher biofilm production in comparison to skin commensals (Fig. 1g). Moreover, in accordance with embp expression, CW20 and CW48 strains showed a significantly high level of binding to human fibronectin (Fig. 1h), whereas all strains showed low ability to attach to collagen (Suppl.

Fig 1).
To assess if S. epidermidis isolates contribute to high non-healing rates of VLUs observed in patients, we used a human ex vivo wound model and evaluated biofilm formation and wound closure upon infection. Immunostaining of S. epidermidis and H&E in wounds infected with CW9 and CW48 showed localization of bacteria in the wound bed, overlying the epidermis, and between the epidermal-dermal junction at the wound edges (Fig. 2a&b). We also detected high bacterial load for CW9 and CW48, while the strain CW20 had limited growth in human ex vivo wounds (Fig. 2a-c), despite its potency to form biofilm in vitro. In contrast to VLU isolates, healthy skin isolates were unable to grow in the ex vivo wounds (Fig. 2c). The potent biofilmforming ability of CW9 and CW48 indicated correlation of their virulence primarily with the strains' ABR, rather than the mere presence of biofilm-associated genes. In addition, CW9 formed a wound biofilm even in the absence of ica operon, aap and sdrF, thought to be indispensable for biofilm formation 11 . Next, we evaluated re-epithelialization in ex vivo wounds 4 days post-infection. While control uninfected wounds showed high levels of wound closure, VLU isolates inhibited healing (Fig. 2b & d) and caused detachment of the epidermis from the dermis which can be associated with Staphylococci proteolytic activity 16 . However, genes encoding cysteine (ecp) and serine (esp) exoproteases were detected among all isolates (Suppl  Fig. 2). We also analyzed the presence of genes encoding the agr system, a virulence factor regulator 17 , and the cassette chromosome recombinase-encoding genes ccrA and ccrB 18 and found no association with strains' biofilm formation (Suppl Fig. 3&4). Next, we examined gene expression of the pro-inflammatory markers and AMPs. Only biofilm formed by CW9 and CW48 elicited a significant induction of IL-1β and IL-8 (Fig. 2e). Infection with CW48 also resulted in increased expression of IL-6, while commensal strain CCN024 was the only inducer of AMP LL-37 (Fig. 2e). Importantly, the ability of VLU isolates to form the biofilm in ex vivo wounds correlated with the healing outcomes in patients with VLU (Fig. 2f). CW9 and CW48 strains were isolated from VLUs with no improvement in healing during 8 weeks of standard of care, which correlates with the biofilm forming ability in the ex vivo wounds (Fig. 2f). In contrast, CW20 isolate which showed low levels of survival in ex vivo wounds, and lack of mupA and mecA, was isolated from an ulcer that healed (Fig. 2f).
Our study revealed the major fitness traits of S. epidermidis isolates from chronic VLU involving ABR, specifically mecA and mupA, while the biofilm formation relied solely on embp and SdrG.
Furthermore, we show that the capability of chronic wound isolates to form biofilms and elicit pro-inflammatory response in human wound model, correlates with the non-healing outcomes in patients with VLU. We also recognize limitations of our study, including a limited number of VLU and commensal strains. Future studies will involve characterization of the strains from a larger number of patients and evaluations of polymicrobial biofilms. Regardless, our data underscores concern of multi-drug resistant (MDR) S. epidermidis in chronic wounds suggesting increased infection risk elsewhere in the body of affected patients. Increasing bacterial resistance among chronic wound isolates and the association with impaired healing necessitates development of novel antimicrobial and antibiofilm approaches aimed to prevent selection of MDR strains.

Patient population
Patients presenting to the wound clinic at the University of Miami with chronic VLUs were recruited after informed consent was obtained under approved IRB protocol (#20180468).
Participants presented with non-infected target ulcers >2 cm 2 and at least one month duration.
Exclusion criteria included use of systemic or topical antibiotics, immunosuppressants, and cellular therapy in the month preceding the study. All subjects received the standard-of-care including a dressing regimen and a 4 layered compression bandage system. Patients were monitored in the wound clinic for 8 weeks or until wound closure was achieved. Tissue that was debrided was collected at the initial visit for bacterial isolation and characterization.

Bacterial strains and growth conditions
Chronic wound isolates of S. epidermidis (CW9, CW20, CW48 strains) were isolated from debridement VLU tissue using a standard microbiology approach 1 . Human commensal S. epidermidis CCN024, and CCN027 strains, were isolated from healthy volunteers and characterized by phenotypic and qPCR identification techniques as previously described 1,2 . All bacteria were preserved with 20% glycerol at -80° C until the time of culture in Tryptic Soy Broth (TSB) at 37°C, overnight.

DNA isolation and shotgun sequencing
DNA from S. epidermidis isolates was extracted using the DNeasy Blood and Tissue Kit (Qiagen) with a modified lysis step. Enzymatic lysis was performed by using lysostaphin (0.5 mg/ml) in 20 mM Tris EDTA buffer supplemented with 1.2% Triton X-100 for 1 hour at 37°C.

Statistical analysis
All data are presented as mean values ± standard deviation (SD). One-way ANOVA followed by Dunnett's post hoc test were used to compare all the isolates relative to selected healthy skin isolate or uninfected control. A p value less than 0.05 was considered statistically significant. The statistical analysis was performed and graphs were prepared using GraphPad Prism 8 software.
Additional methods are provided in Supplementary data.

Acknowledgments
We dedicate this work to late Dr. Gregory Plano. We thank Cheyanne Head for the technical support. We are also grateful to our patients and all current and past members of IP and MT-C laboratories for their overall support.

Data availability statement
The authors declare that all data supporting the findings of this study are available within the article and its supplementary information files or from the corresponding author upon reasonable request. Raw data and analyzed metagenomic data supporting the findings in this study have been deposited under BioProject ID PRJNA906272.

Competing Interests
The Authors declare no competing financial or non-financial interests. of all isolates to fibronectin; data are presented as the mean ± SD from results obtained from three independent experiments (n=6-12). One-way ANOVA followed by Dunnett's post hoc test was used to compare the results of all isolates relative to CCN027 healthy skin strain (*p<0.05, **p<0.01, ***p<0.001).